ABSTRACT The Zika virus (ZIKV) is the cause of an explosive pandemic of infections across South and Central America, the Caribbean, and the southeastern region of the United States. ZIKV is a flavivirus, a family of small positive strand RNA viruses that includes Dengue virus (DENV), West Nile virus (WNV), Yellow fever virus (YFV), and hepatitis C virus (HCV). ZIKV is transmitted to humans by mosquitoes of the Aedes genus. For the most part, ZIKV causes mild symptoms that mimic dengue fever; however, ZIKV infections in the Americas have been associated with congenital microcephaly and to the neurotropic Guillain-Barr syndrome. This prompted the World Health Organization to declare ZIKV to be a ?public health emergency of international concern?. Despite the significant medical need for prophylactic or therapeutic treatments, there are no vaccines or drugs that have been approved for ZIKV. Therefore, novel therapeutic agents for prophylaxis or treatment of acute ZIKV infections are needed. To address this significant unmet medical need, we will utilize a stable ZIKV replicon cell line to carry out a high-throughput screen to identify novel inhibitors of ZIKV replication, which will be developed into drugs for ZIKV infections. Our approach is based on the strong scientific premise that is built on the longstanding success of flavivirus replicons in drug discovery and development. Recently, the laboratory of our collaborator Pei-Yong Shi (University of Texas Medical Branch) reported the construction and validation of a ZIKV replicon cell line (ZIKV Rep-neo). In preliminary studies, we have used the ZIKV Rep-neo cell line to successfully carry out a small-scale screen of FDA-approve compounds, demonstrating the feasibility of our approach. In Phase I, we will apply the ZIKV replicon assay to screen a library of ?350,000 small molecules for compounds that inhibit replication, and will use counter screens and PAINS filters to eliminate non-specific inhibitors and cytotoxic compounds. We will prioritize the hits based on the results of a panel of secondary assays to assess the potency against infectious ZIKV, spectrum of activity against flaviviruses, cytotoxicity, and the drug-like properties of these compounds. The mechanism of action of prioritized compounds will be investigated to verify that prioritized hits target viral proteins, and preliminary structure activity relationships will be assessed. Inhibitors that meet the stringent criteria listed in the milestones for each specific aim will undergo hit to lead optimization in a subsequent Phase II project. In Phase I of this project we will accomplish the following Specific Aims. Aim 1. Identify potent inhibitors of the ZIKV replicon in a high-throughput screen. Aim 2. Prioritize confirmed hits based on potency, specificity, and drug-like properties. Aim 3. Determine the mechanism of action of prioritized inhibitors. Aim 4. Establish preliminary structure activity relationships for prioritized inhibitors.